Alkaloid glycoside for use as a medicament

ABSTRACT

The invention concerns an alkaloid glycoside for use in medicine. In a preferred aspect, the alkaloid glycoside is used for the stimulation of a class I-restricted immune response and/or a class II-restricted immune response. In a preferred aspect, the alkaloid glycoside is tomatine.

[0001] The present invention relates to a compound. More especially, thepresent invention relates to an adjuvant for use in a vaccine. In a moreparticular embodiment, the present invention relates to a glycosideadjuvant.

[0002] In the majority of vaccines used to date antibodies are raisedagainst proteins that are present on the surfaces of disease-causing(pathogenic) organisms (e.g. viruses, bacteria). Vaccine formulationstypically contain inactivated or killed pathogens. In many situations itis desirable to elicit an antibody response against a particular set ofproteins, peptides or carbohydrates that are present in the pathogen.This leads to a well-defined response which can be targeted againstproteins that are highly conserved between different strains of thepathogen, thereby achieving a broader-based immunity.

[0003] Furthermore, the ability to stimulate both class I- and classII-restricted immune responses is deemed to be important because itleads to a more comprehensive level of immunity than is currentlyachieved with existing vaccines.

[0004] Briefly, class II-restricted responses involve the production ofantibodies that are specific to the proteins or peptides used in thevaccine—this is often referred to as the humoral immune response.

[0005] Class I-restricted immune responses are cell-mediated responsesthat are responsible for detecting and dealing with viral infections. Incontrast to the class II-restricted responses, class I responses involvethe activation of CD8+ cytotoxic T lymphocytes (CTLs). These CTLs areactivated by oligomeric peptides that are derived from non-selfproteins. These peptides are presented at the surface of cells inconjunction with the products of the major histocompatibility complex I(MHC-1). Activated CTLs are able to recognise and destroy virus-infectedcells that have non-self oligopeptides on their surfaces.

[0006] The use of adjuvants in vaccine formulations leads to enhancedimmune responses, particularly against soluble proteins and peptideswhich are only weakly immunogenic.

[0007] Several adjuvants have been described in the literature (e.g.complete or incomplete Freund's adjuvant) but most are only ofexperimental interest because of their severe inflammatory side-effects.So far, saponin-based adjuvants are believed by some to be amongst themost promising because of their activity and their relatively lowtoxicity and inflammatory side-effects.

[0008] By way of example, EP-A-0231039 discloses the use of a saponinglycoside as an adjuvant for vaccines. These particular adjuvants, whichare in the form of nanoparticles, are characterised by their uniquearchitectures and are often referred to as immunostimulatory complexes(ISCOMS). These are produced by mixing a saponin glycoside andcholesterol in aqueous solutions, often in the presence of a lipid anddetergent, followed by one or more purification steps. The saponin ispresent in at least its critical micellar concentration (CMC). Theability of ISCOMS to elicit high antibody titres against a wide range ofantigenic proteins and peptides has been demonstrated as has theirability to induce CTL activity (i.e. the ISCOM nanoparticles canstimulate both class I- and class II-restricted immune responses).

[0009] However, there is still a continuing need to develop adjuvantsfor vaccines, in particular adjuvants that can stimulate classI-restricted and/or class II-restricted immune responses to naturallyoccurring or synthetic proteins and oligopeptides.

[0010] According to a first aspect of the present invention there isprovided an alkaloid glycoside for use in medicine.

[0011] According to a second aspect of the present invention there isprovided an alkaloid glycoside for the stimulation of a classI-restricted immune response and/or a class II-restricted immuneresponse.

[0012] According to a third aspect of the present invention there isprovided an alkaloid glycoside for use as a vaccine adjuvant.

[0013] According to a fourth aspect of the present invention there isprovided the use of an alkaloid glycoside in the manufacture of amedicament for use as a vaccine.

[0014] According to a fifth aspect of the present invention there isprovided the use of an alkaloid glycoside as an adjuvant in themanufacture of a medicament for use as a vaccine.

[0015] According to a sixth aspect of the present invention there isprovided the use of an alkaloid glycoside in the manufacture of amedicament to stimulate a class I-restricted immune response and/or aclass II-restricted immune response.

[0016] According to a seventh aspect of the present invention there isprovided a process of preparing a composition comprising admixing anagent capable of inducing an immune response with an alkaloid glycoside,wherein the agent is in an amount to cause an induction of an immuneresponse and wherein the alkaloid glycoside is in an amount for use asan adjuvant.

[0017] According to an eighth aspect of the present invention there isprovided a process of preparing a composition comprising admixing anagent capable of inducing an immune response with an alkaloid glycoside,wherein the agent is in an amount to cause an induction of an immuneresponse and wherein the alkaloid glycoside is in an amount to causestimulation of a class I-restricted immune response and/or a classII-restricted immune response.

[0018] According to a ninth aspect of the present invention there isprovided a method of treatment, the method comprising administering to asubject a composition wherein the composition comprises an agent capableof inducing an immune response within the subject and wherein thecomposition also comprises an alkaloid glycoside, wherein the agent isin an amount to cause an induction of an immune response and wherein thealkaloid glycoside is in an amount for use as an adjuvant.

[0019] According to a tenth aspect of the present invention there isprovided a method of treatment, the method comprising administering to asubject an alkaloid glycoside, wherein the alkaloid glycoside is in anamount to cause stimulation of a class I-restricted immune responseand/or a class II-restricted immune response.

[0020] According to an eleventh aspect of the present invention there isprovided a method of treatment, the method comprising administering to asubject in need of treatment a composition wherein the compositioncomprises an agent capable of inducing an immune response within thesubject and wherein the composition also comprises an alkaloidglycoside, wherein the agent is in an amount to cause an induction animmune response and wherein the alkaloid glycoside is in an amount tocause stimulation of a class I-restricted immune response and/or a classII-restricted immune response.

[0021] According to a twelfth aspect of the present invention there isprovided the combination of an alkaloid glycoside and an agent capableof inducing an immune response.

[0022] According to a thirteenth aspect of the present invention thereis provided the combination of two or more compositions for sequentialor consecutive administration to a subject, wherein at least onecomposition comprises an alkaloid glycoside and at least one othercomposition comprises an agent capable of inducing an immune response.

[0023] Thus, the present invention is based on the surprising findingthat it is possible to use an alkaloid glycoside for a medical usuage,in particular for the stimulation of a class I-restricted immuneresponse and/or a class II-restricted immune response. This finding ishighly surprising as R. Bomford, M. Stapleton, J. E. Beesley, E. AJessup, K. R. Price, G. R. Fenwick (in “Adjuvanticity and ISCOMformation by structurally divests saponins”; Vaccine, vol.10, (1992),572-577.) state that glycoalkaloids (i.e. alkaloid glycosides) lackadjuvant activity.

[0024] The term “subject” includes any one or more different types ofanimal, including humans. Preferably, the term means a human.

[0025] The term “glycoside” as used herein means a derivative of a sugar(which may be a simple sugar or a complex sugar) in which the hydroxylgroup attached to carbon 1 of the sugar is substituted by an alcoholic,or other, aglycone group..

[0026] In a preferred embodiment, the term “glycoside” as used hereinmeans a chemical entity/substance comprising a steroid, triterpene orother polycyclic aliphatic structure and one or more carbohydrates.

[0027] The term “alkaloid glycoside” as used herein means a glycosidecapable of combining with acids to form salts.

[0028] In a preferred embodiment, the term “alkaloid glycoside” as usedherein means a glycoside where the aglycone group is a steroid,triterpene or other polycylic aliphatic structure which contains atleast one nitrogen heterocycle (i.e. a heterocyclic group comprising atleast N).

[0029] Preferably, the nitrogen heterocycle is a derivative ofpyrrolidine, piperidine, imidazoline, piperazine, morpholine, pyridine,quinoline, isoquinoline, or pyrimidine.

[0030] Preferably the alkaloid glycoside is substantially (virtually)water insoluble.

[0031] Preferably, the term “substantially (virtually) water insoluble”means having a solubility of less than 300 mg per 1000 cc H₂O.

[0032] Examples of suitable alkaloid glycosides may include tomatine,chaconine commersonine, demissine, solanine and solasonine.

[0033] For the present invention the alkaloid glycoside may be aspecific alkaloid glycoside or it may be a mixture of alkaloidglycosides.

[0034] Preferably, the alkaloid glycoside is at least tomatine (viz.(3β, 5α, 22β, 25S)-Spirosolan-3-ylO-β-D-glucopyranosyl-(1-2)-O-[β-D-xlopyranosyl-(1-3)]-O-β-D-glucopyranosyl-(1-4)-β-D-galactopyranoside).

[0035] Preferably, the alkaloid glycoside is tomatine.

[0036] In each of the aspects of the present invention the alkaloidglycoside may be used in combination with one or more other adjuvants,such as saponin glycoside.

[0037] In each of the aspects of the present invention the alkaloidglycoside may be used in combination with one or more pharmaceuticallyacceptable carrier(s) and/or diluent(s) and/or excipient(s).

[0038] In each of the aspects of the present invention the alkaloidglycoside may be used in combination with one or more other activeagent(s).

[0039] The composition of the present invention may be used forprophylactic treatment or for curative treatment.

[0040] Preferably, the composition of the present invention may be usedfor prophylactic treatment.

[0041] Preferably, the composition is a vaccine composition.

[0042] Preferably, the composition of the present invention istranslucent.

[0043] Preferably the method of treatment is a method of vaccination orimmunisation.

[0044] In the method of treatment, the alkaloid glycoside may beadministered at—or delivered to—a different site on or in a subject thansome or all of the agent that is capable of inducing an immune response.

[0045] Preferably, the immune response is at least partial vaccinationor immunisation.

[0046] More preferably, the immune response is substantial vaccinationor immunisation.

[0047] In a preferred aspect, the agent capable of inducing an immuneresponse is a protein or oligopeptide.

[0048] Typically, the protein or oligopeptide is isolatable from avirus, a bacterium, a parasite, or an animal cell.

[0049] In a more preferred aspect, the agent capable of inducing animmune response is a naturally occurring protein or oligopeptide or asynthetic protein or oligopeptide.

[0050] Here the term “synthetic” includes proteins or oligopeptides madeby recombinant DNA techniques and/or proteins or oligopeptides made bysynthetic chemical techniques.

[0051] Preferably, the protein or oligopeptide is equivalent to a T cellepitope.

[0052] For some applications, the protein or oligopeptide may be watersoluble.

[0053] Preferably, the term “water soluble” means ” means having asolubility of at least 300 mg per 1000 cc H₂O.

[0054] For some applications, the protein or oligopeptide may be morewater soluble than the alkaloid glycoside.

[0055] More preferably the protein or oligopeptide is weaklyimmunogenic. The term “weakly immunogenic” means that when administeredin the absence of adjuvants, the agent induces less than a tenfoldincrease in antibody titres or less than 45% cell lysis in an assay ofCTL activity.

[0056] For some applications, preferably the protein or oligopeptide issubstantially purified prior to admixture with the alkaloid glycoside.

[0057] With the present invention, the alkaloid glycoside may be used incombination with one or more agents capable of inducing an immuneresponse.

[0058] For some applications, preferably the alkaloid glycoside is usedin combination with one specific type of agent capable of inducing animmune response.

[0059] Preferably, the alkaloid glycoside of the present invention isused to stimulate both class I-restricted and class II-restricted immuneresponses.

[0060] More preferably, the alkaloid glycoside of the present inventionis used to stimulate both class I-restricted and class II-restrictedimmune responses to naturally occurring or synthetic proteins andoligopeptides.

[0061] In the combination aspect of the present invention, the alkaloidglycoside need not necessarily be admixed with the agent capable ofinducing an immune response. Preferably, however, the alkaloid glycosideis admixed with the agent capable of inducing an immune response.

[0062] In a preferred aspect, the alkaloid glycoside may be admixed withan agent that is capable of suppressing the haemolytic activity of thealkaloid glycoside.

[0063] A preferred agent that is capable of suppressing the haemolyticactivity of the alkaloid glycoside is a sterol.

[0064] Preferred sterols that are capable of suppressing the haemolyticactivity of the alkaloid glycoside are sterols of animal or plant originand may include any one or more of cholesterol, stigmasterol,lumisterol.

[0065] A highly preferred agent that is capable of suppressing thehaemolytic activity of the alkaloid glycoside is cholesterol.

[0066] In a preferred aspect, the alkaloid glycoside may be admixed witha solubiliser for the alkaloid glycoside—particularly if the alkaloidglycoside is substantially insoluble in water.

[0067] In a preferred aspect, the solubiliser is a detergent.

[0068] Suitable detergents include any one or more of a non-ionicdetergent, an ionic detergent, and a zwitterionic detergent.

[0069] Preferably, the detergent is at least a non-ionic detergent.

[0070] Preferably, the detergent is a non-ionic detergent.

[0071] A preferred detergent is β-octylglucopyranoside.

[0072] If a detergent is used, then preferably the detergent is removedfrom the composition that is to be used for the method of treatment. Inthis regard, the art is replete with techniques to remove detergents. Byway of example, the detergent is removed by dialysis, chromatography orgradient centrifugation. Preferably, the detergent is removed bydialysis.

[0073] The composition of the present invention may also include othercomponents—such as one or more lipids.

[0074] In a more preferred aspect, the composition of the presentinvention thus comprises tomatine, cholesterol, a non-ionic detergent,and saline. Cholesterol is present to suppress the haemolytic activityof tomatine. Since tomatine is virtually insoluble in water, thenon-ionic detergent is employed to solubilise it. The mixture isdialysed against saline to remove the detergent. In this way theadjuvant formulation is obtained as a translucent dispersion.

[0075] With the present invention, the alkaloid glycoside may beadministered subsequent to, and/or consecutive with, and/or prior toadministration of the agent capable of inducing an immune response.Preferably, at least some of the alkaloid glycoside is administeredconsecutively with at least some of the agent capable of inducing animmune response.

[0076] For some applications, preferably at least a substantialproportion of the alkaloid glycoside is administered consecutively withat least a substantial proportion of the agent capable of inducing animmune response.

[0077] With the present invention the some or all of the alkaloidglycoside may be administered in the same composition as some or all ofthe agent capable of inducing an immune response.

[0078] Alternatively, the alkaloid glycoside may be administered in adifferent composition as the agent capable of inducing an immuneresponse.

[0079] Preferably, the alkaloid glycoside is administered in the samecomposition as the agent capable of inducing an immune response

[0080] The present invention will now be described by way of exampleonly. In the following examples, room temperature was 20° C.

EXAMPLE 1

[0081] {Preparation of composition RAM-1*—which comprises an adjuvantaccording to the present invention and an agent capable of inducing animmune response. }

Part A

[0082] 25 mg tomatine, 3.1 mg phosphatidylethanolamine and 125 mgβ-octylglucopyranoside were added to 4 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 60° C. and shakenvigorously over a two hour period using a vortex mixture until a uniformfaintly cloudy dispersion was obtained. The mixture was allowed to coolto room temperature before use.

Part B

[0083] 6.25 mg cholesterol, 3.1 mg phosphatidylethanolamine and 125.0 mgβ-octylglucopyranoside were added to 3 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 50° C. and shakenvigorously using a vortex mixer until a clear solution was obtained. Themixture was allowed to cool to room temperature before use.

Part C

[0084] 25 mg ovalbumin (OVA) was dissolved in 3 ml of saline (0.9%sodium chloride solution) at room temperature.

[0085] Part A was mixed with Part B and shaken vigorously over a periodof two hours. To this mixture was added Part C followed by vigorousshaking. The mixture was placed in a thermostatted water bath at 35° C.for 12 hours and shaken regularly during this period. After the mixturewas allowed to cool to room temperature it was placed in a dialysis tube(Mr 10000 cut-off) and dialysed against saline for 24 hours to producethe adjuvant formulation RAM-1*.

EXAMPLE 2

[0086] {Preparation of composition RAM-1—which comprises an adjuvantaccording to the present invention (but no agent capable of inducing animmune response). }

Part A

[0087] 25 mg tomatine, 3.1 mg phosphatidylethanolamine and 125 mgβ-octylglucopyranoside were added to 4 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 60° C. and shakenvigorously over a 2 hour period using a vortex mixture until a uniformfaintly cloudy dispersion was obtained. The mixture was allowed to coolto room temperature before use.

Part B

[0088] 6.25 mg cholesterol, 3.1 mg phosphatidylethanolamine and 125.0 mgβ-octylglucopyranoside were added to 3 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 50° C. and shakenvigorously using a vortex mixer until a clear solution was obtained. Themixture was allowed to cool to room temperature before use.

[0089] Part A was mixed with Part B and shaken vigorously over a periodof two hours. To this mixture was added 3 ml of saline (0.9% sodiumchloride solution) followed by vigorous shaking. The mixture was placedin a thermostatted water bath at 35° C. for 12 hours and shakenregularly during this period. After the mixture was allowed to cool toroom temperature it was placed in a dialysis tube (Mr 10000 cut-off) anddialysed against saline for 24 hours to produce the adjuvant formulationRAM-1.

EXAMPLE 3

[0090] {Preparation of composition RAM-2*—which comprises an adjuvantaccording to the present invention and an agent capable of inducing animmune response.}

Part A

[0091] 99 mg tomatine, 12.5 mg phosphatidylethanolamine and 495 mgβ-octylglucopyranoside were added to 4 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 50° C. and shakenvigorously over a two hour period using a vortex mixer until a uniformfaintly cloudy dispersion was obtained. The mixture was allowed to coolto room temperature before use.

Part B

[0092] 25 mg cholesterol, 12.5 mg phosphatidylethanolamine and 495 mgβ-octylglucopyranoside were added to 3 ml of saline (0.9% salinechloride solution). The mixture was warmed to 50° C. and shakenvigorously using a vortex mixer until a clear solution was obtained. Themixture was allowed to cool to room temperature before use.

Part C

[0093] 10 mg ovalbumin (OVA) was dissolved in 3 ml of saline (0.9%sodium chloride solution) at room temperature.

[0094] Part A was mixed with Part B and shaken vigorously over a periodof two hours. To this mixture was added Part C followed by vigorousshaking. The mixture was placed in a thermostatted water bath at 35° C.for 12 hours and shaken vigorously during this period. After the mixturewas allowed to cool to room temperature it was placed in a dialysis tube(Mr 3000 cut-off) and dialysed against saline for 24 hours to producethe adjuvant formulation RAM-2*.

EXAMPLE 4

[0095] {Preparation of composition RAM-2—which comprises an adjuvantaccording to the present invention (but no agent capable of inducing animmune response).}

Part A

[0096] 99 mg tomatine, 12.5 mg phosphatidylethanolamine and 495 mgβ-octylglucopyranoside were added to 4 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 50° C. and shakenvigorously over a two hour period using a vortex mixer until a uniformfaintly cloudy dispersion was obtained. The mixture was allowed to coolto room temperature before use.

Part B

[0097] 25 mg cholesterol, 12.5 mg phosphatidylsethanolamine and 494 mgβ-octylglucopyranoside were added to 3 ml of saline (0.9% sodiumchloride solution). The mixture was warmed to 50° C. and shakenvigorously using a vortex mixer until a clear solution was obtained. Themixture was added to cool to room temperature before use.

[0098] Part A was mixed with Part B and shaken vigorously over a periodof two hours. To this mixture was added 3 ml of saline (0.9% sodiumchloride solution) followed by vigorous stirring. The mixture was placedin a thermostatted water bath at 35° C. for 12 hours and shakenregularly during this period. After the mixture was allowed to cool toroom temperature it was placed in a dialysis tube (Mr 3000 cut-off) anddialysed against saline for 24 hours to produce the adjuvant formulationRAM-2.

ANTIBODY TITRE ASSAYS

[0099] Four sets of experiments were carried out as shown in Table 1.Each group consisted of six mice. TABLE 1 Group I Antigen givensubcutaneously (s.c.) at t = 0 (no adjuvants) Group II RAM-1* or RAM-2*given s.c. at t = 0 Group III RAM-1 or RAM-2 given s.c. at t = 0followed by separate injection or antigen at different site 3 hourslater Group IV RAM-1 or RAM-2 given s.c. at t = 0 (no antigen)

[0100] The immunisation/bleeding regime is shown Table 2 below. TABLE 2Immunisation 1st 2nd 3rd 4th — — Days 0 7 21 35 56 84 Weeks 0 1 3 5 8 12Bleeding (pre) 1 2 3 4 5 6

[0101] The results of the experiments were as follows. Anti-OVA specificantibody titres in the blood serum obtained on day 35 from C57BL/6 miceare shown in Table 3. These titres were measured using standard ELISAprocedures and are expressed as logarithm to the base 10 of the highestdilution giving an optical density (OD) reading of >0.1. The backgroundOD was determined by taking the mean of samples from 6 non-immunizedmice. TABLE 3 GROUP I GROUP II GROUP III GROUP IV log₁₀ antibody 2.7 6.03.3 2.0 titre

[0102] The results showed that all adjuvants lead to high titres ofantigen-specific antibodies during the course of the immunisation.Comparison of the data from the Group II and Group III experimentsindicate that the adjuvant effect can be achieved by independentadministration of antigen and antigen-free adjuvant formulation. None ofthe Group I or IV experiments showed increases in antigen-specificantibody titres.

CYTOTOXICITY ASSAYS

[0103] Cytotoxic activity mediated by T lymphocytes was measured usingthe ovalbumin-transfected EL4 (C57BL/6, H-2b thymoma) monoclonal murinecell line as a target. Untransfected EL4 cells were used as negativecontrols. Spleen cells (3×10⁷) from normal or immunised mice primed 14days earlier were incubated with 1.5×10⁶ irradiated (200 Gy) 15-12 cells(gp160 transfectants) or 3T3 cells (which served as controls) for 5days. Target cells (1×10⁶) were labelled with 100 μCi ⁵¹Cr. Target cellswere washed three times in RPMI 1640 medium, diluted to 2×10⁵ cells/ml,and 50 μ (10⁴) of target cells added to each microtitre well containingantigen or peptide (10 μM) and preincubated for 30 min. Effector cellswere resuspended in complete mdium (CM) to yield a range of effector:target (E:T) ratios. Target cells (10⁴) were then incubated at differentE:T ratios in a total volume of 200 μl of 10% CM for an additional 4 h.Supernatants (100 μl) were collected and counted for ⁵¹Cr release.Spontaneous release and maximal release values were determined byincubating target cells in 10% CM alone or 0.1% Triton X-100. PercentCytotoxicity was calculated using the formula:

[0104] 100×(ER−SR)/(MR−SR)

[0105] where ER is experimental release, SR is spontaneous release andER is maximal release. The results shown are shown in Table 4 andrepresent the means of triplicate determinations. TABLE 4 % of Cytotoxiclysis E:T ratio antigen only alum + anitgen RAM-1* 100:1  45 12 60 50:130 4 40 25:1 19 3 33 12.5:1   — — 6

SUMMARY

[0106] In accordance with the present invention, we have found thatalkaloid glycosides—such as tomatine—are able to elicit a strong classI- and class II-restricted immune response when administered withsoluble proteins.

[0107] Other modifications will be apparent to those skilled in the art.For example, solanine or chaconine may be used instead of or in additionto tomatine. By way of further example, any one or more of: Gp120 fromhuman immunodeficiency virus, nucleoprotein from influenza virus, PIouter membrane protein from Neisseria gonorrhoeae, or 055:B5polysaccharide from Escherichia coli may be used instead of or inaddition to OVA.

1. An alkaloid glycoside for use in medicine.
 2. An alkaloid glycosidefor the stimulation of a class I-restricted immune response and/or aclass II-restricted immune response.
 3. An alkaloid glycoside for use asa vaccine adjuvant.
 4. The use of an alkaloid glycoside in themanufacture of a medicament for use as a vaccme.
 5. The use of analkaloid glycoside in the manufacture of a medicament to stimulate aclass I-restricted immune response and/or a class II-restricted immuneresponse.
 6. A combination of an alkaloid glycoside and an agent capableof inducing an immune response.
 7. A method comprising administering toa subject a composition wherein the composition comprises an agentcapable of inducing an immune response within the subject and whereinthe composition also comprises an alkaloid glycoside, wherein the agentis in an amount to cause an induction of an immune response and whereinthe alkaloid glycoside is in an amount to cause stimulation of a classI-restricted immune response and/or a class II-restricted immuneresponse.
 8. The invention according to any one of the preceding claimswherein the alkaloid glycoside is substantially water insoluble.
 9. Theinvention according to any one of the preceding claims wherein thealkaloid glycoside is at least tomatine.
 10. The invention according toany one of the preceding claims the agent capable of inducing an immuneresponse is a protein or oligopeptide.
 11. The invention according toclaim 10 wherein the protein or oligopeptide is equivalent to a T cellepitope.
 12. The invention according to any one of the preceding claimswherein the agent capable of inducing an immune response is weaklyimmunogenic.
 13. An adjuvant, or a composition, or a use, or a method,or a process, or a combination substantially as described herein.